+ + CCR8 FOXp3 Treg cells as master drivers of immune regulation Yiftah Barshesheta,1,2, Gizi Wildbauma,1, Eran Levya, Alon Vitenshteina, Chika Akinseyeb, Jeremy Griggsb, Sergio A. Lirac, and Nathan Karina,d,3 aDepartment of Immunology, Technion, Haifa 31096, Israel; bGlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, SG1 2NY, United Kingdom; cImmunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and dRappaport Family Institute for Research in the Medical Sciences and Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved April 28, 2017 (received for review January 5, 2017) + The current study identifies CCR8 regulatory T cells (Treg cells) as showed in a graft versus host disease (GVHD) model, donor Treg drivers of immunosuppression. We show that in human peripheral cells lacking CCR8 were severely impaired in their ability to blood cells, more than 30% of Treg up-regulate CCR8 following ac- prevent lethal GVHD (31). However, the underlying mechanisms tivation in the presence of CCL1. This interaction induces STAT3- of such observations remained unclear. dependent up-regulation of FOXp3, CD39, IL-10, and granzyme B, The current study uncovers the mechanistic basis by which the resulting in enhanced suppressive activity of these cells. Of the four CCR8–CCL1 axis potentiates Treg cells, its relevance to human human CCR8 ligands, CCL1 is unique in potentiating Treg cells. The biology, and explores the clinical implications of these findings relevance of these observations has been extended using an exper- using an experimental autoimmune disease of the central ner- imental model of multiple sclerosis [experimental autoimmune en- vous system (CNS) (32). cephalomyelitis, (EAE)] and a stabilized version of mouse CCL1 (CCL1–Ig). First, we identified a self-feeding mechanism by which Results CCL1 produced by Treg cells at an autoimmune site up-regulates Of the Four CCR8 Ligands, CCL1 Is Unique in Potentiating the Suppressive the expression of its own receptor, CCR8, on these cells. Adminis- Function of Human Treg Cells. Human CCR8 has four known ligands: tration of CCL1–Ig during EAE enhanced the in vivo proliferation of CCL1, CCL8, CCL16, and CCL18 (33). First we examined whether + these CCR8 regulatory cells while inducing the expression of CD39, one or more of the four human CCR8 ligands may enhance the granzyme B, and IL-10, resulting in the efficacious suppression of suppressive activity of human Treg cells. Fig. 1 summarizes data – ongoing EAE. The critical role of the CCL1 CCR8 axis in Treg cells was obtained from 10 different healthy donors, indicating that of the − − further dissected through adoptive transfer studies using CCR8 / four human CCR8 ligands, CCL1 was unique in potentiating the + mice. Collectively, we demonstrate the pivotal role of CCR8 Treg suppressive function of human Treg cells, detected as an enhanced cells in restraining immunity and highlight the potential clinical im- suppression of Teff proliferation (Fig. 1 A–C). Because CCR8 is plications of this discovery. considered to be the exclusive receptor for CCL1 (26), we sought to confirm that these effects were achieved via CCR8 signaling. As EAE | chemokine | FOXp3 | CCL1 | CD39 shown in Fig. 1D, antihuman CCR8 blocking mAb abolished CCL1-mediated effects in this assay. + wo major populations of CD4 regulatory T cells (Treg cells), Independently, each human CCR8 ligand was tested in a fluo- defined by whether they express the forkhead box protein rometric imaging plate reader (FLIPR) assay (34) configured to T + 3 transcription factor (FOXp3), are thought to play a key role in the detect the induction of intracellular Ca2 flux in response to CCR8 + − maintenance of self-tolerance (1–8). Both FOXp3 and FOXp3 activation in CHO-K1 cells overexpressing human CCR8. As subtypes participate in the regulation of inflammatory autoimmu- shown in Fig. S1, CCL1 induced a dose-dependent up-regulation of nity and in the maintenance of self-tolerance by various mecha- nisms, including regulating the biological function of effector Significance + + − TH1 and TH17 CD4 Tcells(6,7,9–12). CD4 FOXp3 regula- + tory T cells can be categorized as T regulatory-1 cells (Tr1), which The current study identifies CCR8 regulatory T cells (Treg cells) primarily produce IL-10 (12–15), and Th3, which express high levels as drivers of immunosuppression and provides compelling ev- of TGFβ (16). idence of a self-feeding mechanism by which, at an autoim- ∼ – + Chemokines are small ( 8 14 kDa) secreted proteins, structur- mune site, CCL1 produced by FOXp3 Treg cells upregulates the ally similar to cytokines, that regulate cell trafficking through in- expression of its own receptor, CCR8, on these cells, and po- teractions with a subset of seven transmembrane G protein-coupled tentiates their in vivo proliferation and suppressive activities as receptors (GPCRs) (17), and many of them are associated with driver Treg cells. The suppression of ongoing autoimmunity by a chemotaxis of leukocytes to inflammatory sites (18–20). Almost 15 y stabilized version of the chemokine (CCL1–Ig) highlights the agoweshowedthat,inadditiontotheir role in chemoattraction, translational potential of these findings. + chemokines are also involved in directing effector CD4 T-cell Author contributions: Y.B., G.W., J.G., and N.K. designed research; Y.B., G.W., E.L., A.V., (Teff) polarization, by showing that the CXCR3 ligand CXCL10 directs the lineage development of TH1 cells (21, 22). C.A., and J.G. performed research; S.A.L. contributed new reagents/analytic tools; and N.K. wrote the paper. More recently we identified two different chemokines that are Conflict of interest statement: N.K., Y.B., and G.W. hold a pending patent on CCL1-based involved in the lineage development of Tr1 cells (23, 24). The + therapy of autoimmunity and graft-versus-host disease that has been outlicensed current study focuses on FOXp3 Treg cells and on the interplay to GlaxoSmithKline. between CCR8 and its ligands. This article is a PNAS Direct Submission. In mouse, the chemokine receptor CCR8 is expressed princi- 1Y.B. and G.W. contributed equally to this work. pally on Treg cells and also notably on small fractions of TH2 cells, 2Present address: Faculty of Medicine in The Galilee, Bar-Ilan University, Safed 1311502, monocytic cells, and NK cells (25–28), but not TH1 cells (29, 30). Israel. A similar expression pattern is seen in humans, in which CCR8 is 3To whom correspondence should be addressed. Email: [email protected]. ∼ + additionally found on 2% of CD8 cells (30). CCR8 is known to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. be critical for Treg function. For example, Coghill et al. recently 1073/pnas.1621280114/-/DCSupplemental. 6086–6091 | PNAS | June 6, 2017 | vol. 114 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1621280114 Downloaded by guest on October 1, 2021 setup and showed preferential early transcription of CCR8 and A 100000 B * FOXp3, as discussed later (Fig. S2). SE) 80000 * P<0.001 A subsequent set of experiments was conducted to deter- 60000 mine whether CCL1 might also increase FOXp3 expression in + − high − 40000 CD4 CD25 CD127 T cells (FOXp3 ) to convert them into + 20000 FOXp3 . We could not find compelling evidence to show that − + 0 CCL1 may directly convert FOXp3 T cells into FOXp3 (Fig. 2E). CFSE Taken together, these data imply that CCL1 potentiates the + Proliferation (cpm Proliferation * P<0.001 suppressive activity of CCR8 Treg cells by inducing FOXp3, D C Teff CD4+CD127-CD25+ 20000 Teff+Treg A B No CCL1 + CCL1 16000 * P<0.01 7 PBS CCL1 (100ng/ml) 12000 ** 6 CD4 ** 8000 5 *P<0.001 4 4000 * *P<0.001 3 * CCR8 Thymidine uptake (CPM) 0 3 2 CD4+CD127+CD25- H 1 No CCL1 + CCL1 0 Relative expression (AU) Relative expression CD4 + + CCR8 Fig. 1. CCL1 selectively potentiates suppressive function in CD4 CD25 C Before sorting After sorting of CD25+CD127 low CD127low T cells. (A–C) CCL1 potentiates the suppressive function of human + + low CD4 CD25 CD127 T cells. Freshly isolated human Treg cells were activated CD4 CD4 in the presence of each of the known CCR8 ligands used in suppression assay (SI *P<0.01 3 Methods). Proliferation of Teff cells was detected by incorporation of [ H] thy- midine incorporation (results in A areshownasmeanoftriplicates± SE), or by FOXp3 FOXp3 CFSE staining of effector T cells (B and C). B shows the results of a representative experiment. C summarizes CFSE results of all 10 healthy donors as a scattered D no chemokine CCL1 plot (P < 0.001 unpaired Student’s t test). (D) CCL1 directs its function via CCR8. Suppression assays were conducted as described for A above, with or without *P<0.0001 FOXp3 addition of an anti-CCR8 blocking mAb. Results of one of three independent experiments are shown as mean of triplicates ± SE. Significance was determined CD39 by two-tailed unpaired Student’s t test (*P < 0.01). FOXp3 *P<0.0001 2+ Ca flux in response to CCR8 activation, whereas no effect was Granzyme B seen in the presence of CCL8, CCL16, or CCL18. We note that these data differ from a publication showing that CCL18 may also + FOXp3 induce Ca2 flux via CCR8 (33). P<0.0001 Collectively, these data show that of the four CCR8 ligands, IL-10 + E No chemokine CCL1 TGF- only CCL1 induces Ca2 flux and potentiates the suppressive activity of these cells. FOXp3 CCL1 Potentiates Human Treg Cells by Inducing CCR8, FOXp3, CD39, Granzyme B, and IL-10 Expression. At 36 h postactivation of cultured CD4 human Treg cells that were, or were not, supplemented with CCL1, Fig. 2. CCL1 potentiates human Treg cells by inducing CCR8, FOXp3, CD39, they were examined for the transcription of various genes known to granzyme B, and IL-10 expression.